Target Detection with Nanopore

ABSTRACT

Provided are methods for detecting a target molecule or particle suspected to be present in a sample, comprising (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore comprising an opening in a structure that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule or particle bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2014/046397, filed Jul. 11, 2014, which claims the benefit of PCTApplication No. PCT/US2014/036861, filed May 5, 2014, the contents ofwhich are each incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 20, 2016, isnamed 34004US_CRF_sequencelisting.txt and is 563 bytes in size.

BACKGROUND

Detection of nano-scale and micro-scale particles, such as circulatingtumor cells, bacteria and viruses, has immense clinical utility.Currently available methods include immunohistochemistry and nucleicacid-based detection, and cell proliferation is typically requiredbefore a sensitive detection can be carried out.

Molecular detection and quantitation are also important, and can becarried out with various methods depending on the type of the molecule.For instance, a nucleotide sequence can be detected by virtue of itssequence complementarity to a probe or primer, through hybridizationand/or amplification, or in fewer occasions, with a protein thatrecognizes the sequence. A protein, on the other hand, is commonlydetected with an antibody that specifically recognizes and binds theprotein. An enzyme-linked immuno sorbent assay (ELISA), in this respect,is highly commercialized and commonly used.

Methods also exist for detecting or quantitating various other large orsmall molecules, such as carbohydrates, chemical compounds, ions, andelements.

Methods and systems for highly sensitive detection of molecules as wellas particles, such as tumor cells and pathogenic organisms, have broadapplications, in particular, clinically, for pathogen detection anddisease diagnosis, for instance. Additionally, such detection may: allowfor the personalization of medical treatments and health programs;facilitate the search for effective pharmaceutical drug compounds andbiotherapeutics; and enable clinicians to identify abnormal hormones,ions, proteins, or other molecules produced by a patient's body and/oridentify the presence of poisons, illegal drugs, or other harmfulchemicals ingested or injected into a patient.

Currently available techniques for the detection of molecules andparticles are generally expensive, labor-intensive, skill-intensive,and/or time-intensive. A need exists for improved detection techniques,which produce accurate results quickly, cheaply, and easily.

SUMMARY

Various aspects disclosed herein may fulfill one or more of theabove-mentioned needs. The systems and methods described herein eachhave several aspects, no single one of which is solely responsible forits desirable attributes. Without limiting the scope of this disclosureas expressed by the claims that follow, the more prominent features willnow be discussed briefly. After considering this discussion, andparticularly after reading the section entitled “Detailed Description,”one will understand how the sample features described herein provide forimproved systems and methods.

In one embodiment, the present disclosure provides a method for assayingwhether a target molecule or particle is present in a sample, the methodcomprising: (a) contacting the sample with (i) a fusion moleculecomprising a ligand capable of binding to the target molecule orparticle and a binding domain, and (ii) a polymer scaffold comprising atleast one binding motif to which the binding domain of the fusionmolecule is capable of binding, under conditions that allow the targetmolecule or particle to bind to the ligand and the binding domain tobind to the binding motif; (b) loading the polymer into a devicecomprising a pore that separates an interior space of the device intotwo volumes, and configuring the device to pass the polymer through thepore from one volume to the other volume, wherein the device comprises asensor configured to identify objects passing through the pore; and (c)determining, with the sensor, whether the fusion molecule bound to thebinding motif is bound to the target molecule or particle, therebydetecting the presence or absence of the target molecule or particle inthe sample.

In some aspects, the target molecule is selected from the groupconsisting of a protein, a peptide, a nucleic acid, a chemical compound,a lipid, a receptor, an ion, and an element.

In some aspects, the target particle is selected from the groupconsisting of protein complexes and protein aggregates, peptideaggregates, protein/nucleic acid complexes, fragmented or fullyassembled viruses, bacteria, cells, and cellular aggregates.

In some aspects, step (a) of the method for assaying whether a targetmolecule or particle is present in a sample is performed prior to step(b). In some aspects, step (b) is performed prior to step (a).

In some aspects, the method further comprises applying a conditionsuspected to alter the binding between the target molecule or particleand the ligand, and carrying out the determination again. In someaspects, the condition is selected from the group consisting of removingthe target molecule or particle from the sample, adding an agent thatcompetes with the target molecule or particle or the ligand for binding,and changing the pH, salt concentration, or temperature.

In some aspects, the binding motif comprises a chemical modification forbinding to the binding domain. In some aspects, the chemicalmodification is selected from the group consisting of acetylation,methylation, summolation, glycosylation, phosphorylation, and oxidation.

In some aspects, the polymer comprises a deoxyribonucleic acid (DNA), aribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA/RNA hybrid,or a polypeptide. In some aspects, the polymer is a synthetic scaffold.

In some aspects, the binding domain is selected from the groupconsisting of a helix-turn-helix, a zinc finger, a leucine zipper, awinged helix, a winged helix turn helix, a helix-loop-helix and anHMG-box.

In some aspects, the binding domain is selected from the groupconsisting of locked nucleic acids (LNAs), PNAs, transcriptionactivator-like effector nucleases (TALENs), clustered regularlyinterspaced short palindromic repeats (CRISPRs), peptides, dendrimers,and aptamers (DNA and/or protein).

In some aspects, the ligand is a protein. In some aspects, the ligand isselected from the group consisting of an antibody, an antibody fragment,an epitope, a hormone, a neurotransmitter, a cytokine, a growth factor,a cell recognition molecule, a nucleic acid, a peptide, and a receptor.In some aspects, the ligand is an aptamer (e.g., DNA, protein, orDNA/protein). In some aspects, the ligand is a small molecule compound.

In some aspects, the binding domain and the ligand are linked via acovalent bond, a hydrogen bond, an ionic bond, a metallic bond, van derWalls force, a hydrophobic interaction, or a planar stackinginteraction, or are translated as a continuous polypeptide, to form thefusion molecule.

In some aspects, the method further comprises contacting the sample witha detectable label capable of binding to the target molecule, targetparticle or target/ligand complex.

In some aspects, the polymer comprises at least two units of the bindingmotif.

In some aspects, the polymer comprises at least two different bindingmotifs. In such aspects, the sample is in contact with at least twofusion molecules, each of which comprises a different binding domaincapable of binding to a different one of the at least two differentbinding motifs, and a different ligand capable of binding to a differenttarget molecule or particle; and the sensor is configured to identifywhether the fusion molecule bound to each binding motif is bound to atarget molecule or particle.

In some aspects, the sensor comprises electrodes further configured toapply a voltage across the two volumes.

In some aspects, the device comprises an upper chamber, a middle chamberand a lower chamber, wherein the upper chamber is in communication withthe middle chamber through a first pore, and the middle chamber is incommunication with the lower chamber through a second pore.

In one aspect, the first pore and second pore are about 1 nm to about100 nm in diameter. Such pores can be suitable for detecting moleculessuch as proteins and nucleic acids. In one aspect, the first pore andsecond pore are as large as about 50,000 nm in diameter, which can besuitable for detecting larger particles such as tumor and bacterialcells.

In some aspects, the pores are about 10 nm to about 1000 nm apart fromeach other. In some such aspects, the distance between the pores issized such that the polymer scaffold may simultaneously extend throughboth the first and second pores. In other aspects, the pores are morethan 1000 nm apart from each other.

In some aspects, each of the chambers comprises an electrode forconnecting to a power supply.

In some aspects, the method further comprises moving the polymer in areverse direction after the binding motif passes through at least onepore, such as to identify, again, whether the fusion molecule bound toeach binding motif is bound to a target molecule or particle.

Also provided are kits, packages or mixtures that detect the presence ofa target molecule or particle. In some aspects, the kit, package ormixture is comprised of (a) a fusion molecule, which itself is a ligandcapable of binding to the target molecule or particle and a bindingdomain, (b) a polymer scaffold, which is comprised of at least onebinding motif to which the binding domain is capable of binding, (c) adevice, which is comprised of a pore that separates an interior space ofthe device into two volumes, wherein the device is configured to allowthe polymer to pass through the pore from one volume to the othervolume, and wherein the device is further comprised of a sensorconfigured to identify whether the binding motif is (i) bound to thefusion molecule while the ligand is bound to the target molecule orparticle, (ii) bound to the fusion molecule while the ligand is notbound to the target molecule or particle, or (iii) not bound to thefusion molecule. In some aspects, the device is further comprised of asecond pore that further separates the interior space of the device suchthat three volumes: an upper chamber, a middle chamber, and a lowerchamber, are present.

In some aspects, the kit, package or mixture further comprises a samplesuspected of containing the target molecule or particle. In someaspects, the sample further comprises a detectable label capable ofbinding to the target molecule, particle, ligand/target complex, orligand/particle complex.

BRIEF DESCRIPTION OF THE DRAWINGS

Provided as embodiments of this disclosure are drawings that illustratefeatures by exemplification only, and not limitation.

FIG. 1 illustrates the detection of a target molecule or particle withone embodiment of the presently disclosed method.

FIG. 2 provides the illustration of a more specific example, where adouble-stranded DNA is used as the polymer scaffold, and a humanimmunodeficiency virus (HIV) envelope protein is used as the ligand. Thecombination is used to detect an anti-HIV antibody.

FIG. 3A, FIG. 3B, and FIG. 3C show representative and idealized currentprofiles of three example molecules, demonstrating that binding betweena target molecule (or particle) and a fusion molecule can be detectedwhen passing through a nanopore, since it has a different currentprofile, compared to that of the fusion molecule alone or the DNA alone.Specifically, FIG. 3A shows current profiles consistent with higher saltconcentrations (>0.4 M KCl, for example at 1M KCl) in the experimentalbuffer and a positive applied voltage, generating a positive currentflow through the pore. By another example, FIG. 3B shows currentprofiles consistent with lower salt concentrations (>0.4 M KCl, forexample at 100 mM KCl) in the experimental buffer and again at apositive applied voltage. By another example, FIG. 3C shows currentprofiles consistent with lower salt concentrations (>0.4 M KCl, forexample at 100 mM KCl) in the experimental buffer and a negative appliedvoltage.

FIG. 4 illustrates the multiplexing capability of the present technologyby including different binding motifs in the polymer scaffold. Suchmultiplexing can be accomplished with one nanopore or more than onenanopore.

FIG. 5A, FIG. 5B, and FIG. 5C illustrate a nanopore device with at leasttwo pores separating multiple chambers.

Specifically, FIG. 5A is a schematic of a dual-pore chip and adual-amplifier electronics configuration for independent voltage control(V₁ or V₂) and current measurement (l₁, or l₂) of each pore. Threechambers, A-C, are shown and are volumetrically separated except bycommon pores.

FIG. 5B is a schematic where electrically, V₁ and V₂ are principallyapplied across the resistance of each nanopore by constructing a devicethat minimizes all access resistances to effectively decouple l₂ and l₂.

FIG. 5C depicts a schematic in which competing voltages are used forcontrol, with arrows showing the direction of each voltage force.

FIGS. 6A, 6B, and 6C illustrate a nanopore device having one poreconnecting two chambers and example results from its use. Specifically,FIG. 6A depicts a schematic diagram of the nanopore device. FIG. 6Bdepicts a representative current trace showing a blockade eventresulting from the passage of a double-stranded DNA passing through thepore. The current amplitude shift amount (Δl=l₀−l_(B)) and duration toare used to quantify the passage event. FIG. 6C depicts a scatter plotshowing the change in current amount (Δl) vs. translocation time (t_(D))for all blockade events recorded over 16 minutes.

FIG. 7A and FIG. 7B depict current traces measured within one embodimentof a nanopore device fabricated in accordance with the presentinvention. The provided current traces show that unbound dsDNA causescurrent enhancement events at KCl concentrations below 0.4 M. Currentenhancements appeared as downward shifts in the provided experiment,since the voltage and current are both negative (as in FIG. 3C).Specifically, in DNA alone control experiments using a 10-11 nm diameterpore in 0.1M KCl at −200 mV, 5.6 kb dsDNA scaffold (FIG. 7A) causesbrief current enhancement events that are 50-70 pA in amplitude and10-200 microseconds in duration. Likewise, 48 kb Lambda DNA (FIG. 7B)causes current enhancement events 50-70 pA in amplitude and 50-2000microseconds in duration.

FIG. 8 depicts a schematic diagram of a polymer scaffold. Specifically,FIG. 8 shows a 5,631 by dsDNA scaffold and the location of 10 total VspRbinding sites. Of the 10 VspR binding sites, 5 are of one 14 base-pairsequence, 3 of a different 18 base pair sequence, and 2 are of a 27 basepair sequence. Also shown are the distances (in base pairs) between thebinding sites.

FIG. 9A and FIG. 9B each show schematic representations of embodimentsof a nanopore with a scaffold passing therethrough. Each also shows aresultant current profile associated with the scaffold passage asmeasured by one embodiment of the disclosed nanopore device. Inparticular, FIG. 9A and FIG. 9B compare events with DNA scaffold alone(FIG. 9A) and VspR-bound DNA (FIG. 9B). Specifically, FIG. 9A shows agraphic depicting the 5,631 by dsDNA scaffold passing through the pore,and a representative current enhancement event (downward 50 pA shiftlasting 100 microseconds) when the scaffold passes through the pore.FIG. 9B shows a graphic depicting multiple VspR bound to a dsDNAscaffold that is passing through the pore, and a representative currentattenuation event (upward 150 pA shift lasting 1.1 milliseconds) whenthe VspR-bound scaffold passes through the pore. At an applied voltageof −100 mV, the open channel current is negative, so downward eventscorrespond to current enhancement events, and upward events correspondto current attenuation events (as in FIG. 3C). The shift direction ispreserved, even though the baseline is zeroed for display purposes.

FIG. 10 shows ten more representative current attenuation eventsdepicted in a current profile consistent with the VspR-bound scaffoldpassing through the pore. All shifts are consistent with currentattenuations; the baseline is zeroed for display purposes.

FIG. 11 shows two representative current events depicted in a currentprofile captured in an experiment with 5.6 kb dsDNA scaffold and RecAprotein at 180 mV and 1M KCl using a 16-18 nm diameter nanopore. Thefirst event is consistent with an unbound dsDNA or possibly a free RecA(or multiple associated RecA proteins) passing through the pore, at 280pA mean current attenuation lasting 70 microseconds. The second event isconsistent with RecA-bound scaffold passing through the pore, at 1.1 nAmean current attenuation lasting 2.7 milliseconds. RecA-bound eventscommonly display deeper blockades with longer duration.

FIG. 12 depicts four more current profiles, each showing arepresentative current event consistent with RecA-bound scaffold passingthrough the pore.

FIG. 13 shows scatter plots and histograms depicting all 1385 eventsrecorded over 10 minutes in one experiment conducted using embodimentsof methods described herein. In the depicted graphs, one data point isprovided for each event. In particular, the depicted graphs show: (a)maximum conductance in nS (maximum current shift in pA divided byvoltage in mV) vs. time duration in seconds, with time duration on alog-scale; (b) a probability histogram of the maximum conductance shiftvalues; (c) mean conductance (mean current shift divided by voltage) vs.time duration, with time duration on a log-scale; (d) a probabilityhistogram of the mean conductance values; and (e) a probabilityhistogram of the time duration on a log-scale.

FIG. 14A, FIG. 14B, and FIG. 14C illustrate results from a nanoporedevice detecting DNA/RecA complexes and RecA-antibody on DNA/RecAcomplexes, and the results differentiating these complexes from unboundDNA and also from free RecA.

Specifically, FIG. 14A is a gel shift assay. Specifically, theDNA/RecA/mAb ARM191 Gel Shift Experiments (EMSA) have lanes: 1) Ladder,top rung 5000 bp; 2) Scaffold DNA only in RecA labeling buffer; 3)DNA/RecA complex, 1:1 RecA protein to theoretical RecA binding sites; 4)DNA/RecA/Ab complex, DNA/Rec incubated with a 1:2000 dilution ofmonoclonal Ab ARM191; 5) Scaffold DNA only in Ab labeling buffer; and 6)Scaffold DNA mixed with mAb (ARM191).

FIG. 14B shows representative events for DNA (230 pA, 0.1 ms), DNA/RecA(390 pA, 1.1 ms), and probable DNA/RecA/Ab (860 pA, 1.5 ms). RecA-boundDNA event amplitudes are uniformly smaller than in earlier figures(FIGS. 11-13) since the pore used to measure these events isconsiderably larger (27-29 nm in diameter).

FIG. 14C depicts a (i) Scatter plot of lΔ/l vs. t_(D) and (ii)horizontal probability histogram of lΔ/l for two separate experimentsoverlaid. In a RecA alone control experiment, 0.5 uM RecA (*) wasmeasured at 180 mV in 1M KCl with a 20 nm diameter pore, generating 767events over 10 min. Note that only 0.6% of RecA events exceed a criteriaof (600 pA, 0.2 ms) under these conditions. In another experiment, threereagents were added in sequence in 1M LiCl. First, 0.1 uM DNA (□) wasmeasured at 200 mV with a 20 nm diameter pore, generating 402 events at0.1 events/sec. After the pore enlarged to 27 nm, 1.25 nM DNA/RecA (•)was added, generating 3387 events at 1.44 events/sec. Lastly, 1.25 nMDNA/RecA/Ab (◯) was added generating 4953 events at 4.49 events/sec.Events exceeding the (600 pA, 0.2 ms) criteria grew monotonically from0% with DNA alone, to 5.2% (176) with DNA/RecA added, and up to 9.8%(485) with DNA/RecA/Ab added. While RecA could have increased eventdurations in LiCl, as shown for DNA, event amplitudes are unlikely toshift significantly toward the (600 pA, 0.2 ms) criteria.

FIG. 15A and FIG. 15B illustrates schematic diagrams of polymerscaffolds consistent with embodiments of the present disclosure.Specifically, FIG. 15A shows a 5.6 kb dsDNA scaffold used in variousexperiments, the scaffold having been engineered to bind 12-merpeptide-nucleic-acid (PNA) molecules, with each PNA having 3biotinylated sites for binding avidin (e.g., neutravidin, and ormonovalent streptavidin). Also shown is FIG. 15B identifying the 25distinct PNA binding sites on the scaffold that localize up to 75 avidinbiomarker binding sites.

FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D illustrate: (FIG. 16A) aschematic of the 5.6 kb dsDNA scaffold passing through a nanopore; (FIG.16B) a schematic of a free neutravidin passing through a nanopore; (FIG.16C) a schematic of the dsDNA labeled with a PNA passing through ananopore, the PNA having all three biotin sites bound by Neutravidin;and (FIG. 16D) corresponding current traces as measured in the chamberabove the pore in a nanopore device fabricated in accordance with thepresent disclosure. In FIG. 16D, the current traces depictrepresentative translocation events in the recorded current fromseparate nanopore experiments with DNA alone, Neutravidin alone, andDNA/PNA/Neutravidin complexes. As detailed in the examples, the deeperand longer event pattern in the D/P/N experiment is identified as aDNA/PNA/Neutravidin event and is clearly distinguished from DNA alone orNeutravidin alone events.

FIG. 17A, FIG. 17B, and FIG. 17C illustrate: (FIG. 17A) a scatter plotof the current shift vs. the duration (lΔ/l vs. t_(D)) of all recordedevents for three separate experiments at 200 mV with 10

11 nm diameter pores, the experiments including: (D)—5.6 kb dsDNA aloneat 1 nM yielding 1301 events over 16 minutes; (N) Neutravidin alone at80 nM yielding 2589 events over 11 minutes; and (D/P/N) DNA/PNA/Neutcomplexes at 60 pM yielding 4198 events over 7.3 minutes. D/P/Nsubpopulations overlap with the N and D control experiment populations,with most events in the DPN experiment matching unbound N eventcharacteristics; (FIG. 17B) a horizontal probability histogram of lΔ/lfor the three data sets. The inset shows a histogram for a subset of 578DPN events with t_(D)>0.08 ms, which attempts to trim out non-DNA eventsfrom the D/P/N data set (from controls, 8% of N events and 54% of Devents have to >0.08 ms). Such DPN events have significant spread inlΔ/l, with 252 (6% of the total) of these longer-duration events above2.4 nA, whereas from controls, only 18 (0.7%) N events and 33 (2.5%) Devents have (lΔ/l, t_(D))>(2.4 nA, 0.08 ms); and (FIG. 17C)DNA/PNA/Neutravidin Gel Shift Experiments (EMSA) with lanes: 1) sizingLadder, top rung 5000 bp; 2) DNA only in labeling buffer; 3) DNA/PNA+3×excess Neut to biotin; 4) DNA/PNA+7× excess Neut to biotin; 5)DNA/PNA+16× excess Neut to biotin; 6) DNA/PNA+36× excess Neut to biotin;and 7) DNA/PNA in labeling buffer.

Some or all of the figures are schematic representations forexemplification; hence, they do not necessarily depict the actualrelative sizes or locations of the elements shown. The figures arepresented for the purpose of illustrating one or more embodiments withthe explicit understanding that they will not be used to limit the scopeor the meaning of the claims that follow below.

DETAILED DESCRIPTION

Throughout this application, the text refers to various embodiments ofthe present devices, compositions, systems, and methods. The variousembodiments described are meant to provide a variety of illustrativeexamples and should not be construed as descriptions of alternativespecies. Rather, it should be noted that the descriptions of variousembodiments provided herein may be of overlapping scope. The embodimentsdiscussed herein are merely illustrative and are not meant to limit thescope of the present invention.

Also throughout this disclosure, various publications, patents andpublished patent specifications are referenced by an identifyingcitation. The disclosures of these publications, patents and publishedpatent specifications are hereby incorporated by reference into thepresent disclosure in their entireties.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an electrode” includes a plurality ofelectrodes, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thesystems, devices, and methods include the recited components or steps,but not excluding others. “Consisting essentially of” when used todefine systems, devices, and methods, shall mean excluding othercomponents or steps of any essential significance to the combination.“Consisting of” shall mean excluding other components or steps.Embodiments defined by each of these transition terms are within thescope of this invention.

All numerical designations, e.g., distance, size, temperature, time,voltage and concentration, including ranges, are approximations whichare varied (+) or (−) by increments of 0.1. It is to be understood,although not always explicitly stated that all numerical designationsare preceded by the term “about”. It also is to be understood, althoughnot always explicitly stated, that the components described herein aremerely exemplary, and that equivalents of such are known in the art.

As used herein, “a device comprising a pore that separates an interiorspace” shall refer to a device having a pore that comprises an openingwithin a structure, the structure separating an interior space into morethan one volume or chamber.

Molecular Detection

The present disclosure provides methods and systems for moleculardetection and quantitation. In addition, the methods and systems canalso be configured to measure the affinity of a molecule binding withanother molecule. Further, such detection, quantitation, and measurementcan be carried out in a multiplexed manner, greatly increasing itsefficiency.

FIG. 1 provides an illustration of one embodiment of the disclosedmethods and systems. More specifically, the system includes a ligand 104that is capable of binding to a target molecule 105 to be detected orquantitated. The ligand 104 can be part of, or be linked to, a bindingmoiety (referred to as “binding domain”) 103 that is capable of bindingto a specific binding motif 101 on a polymer scaffold 109. Together, theligand 104 and the binding domain 103 form a fusion molecule 102. Invarious embodiments, both components of the fusion molecule 102 (i.e.,both the ligand 104 and the binding domain 103) bind to their respectivetargets (e.g., target molecule 105 and binding motif 101, respectively)with high affinity and specificity.

Therefore, if all are present in a solution, the fusion molecule 102binds, on one end, to a polymer scaffold (or simply, “polymer”) 109through the specific recognition and binding between the binding motif101 and the binding domain 103, and on the other end, to the targetmolecule 105 by virtue of the interaction between the ligand 104 and thetarget molecule 105. Such bindings cause the formation of a complex thatincludes the polymer 109, the fusion molecule 102 and the targetmolecule 105.

The formed complex can be detected using a that includes a nanopore (orsimply, pore) 107, and a sensor. The pore 107 is a nano-scale ormicro-scale opening in a structure separating two volumes. The sensor107 may be positioned within or adjacent the pore 107 or elsewherewithin the two volumes. The sensor is configured to identify objectspassing through the pore 107. For example, in some embodiments, thesensor identifies objects passing through the pore 107 by detecting achange in a measurable parameter, wherein the change is indicative of anobject passing through the pore 107. This device is referred throughoutas a “nanopore device”. In some embodiments, the nanopore device 108includes means, such as electrodes connected to power sources, formoving the polymer 109 from one volume to another, across the pore 107.As the polymer 109 can be charged or be modified to contain charges, oneexample of such means generates a potential or voltage across the pore107 to facilitate and control the movement of the polymer 109. In apreferred embodiment, the sensor comprises a pair of electrodes, whichare configured to both detect the passage of objects, and provide avoltage, across the pore 107. In this embodiment, a voltage-clamp or apatch-clamp is used to simultaneously supply a voltage across the poreand measure the current through the pore.

When a sample that includes the formed complex is loaded in the nanoporedevice 108, the nanopore device 108 can be configured to pass thepolymer 109 through the pore 107. When the binding motif 101 is withinthe pore or adjacent to the pore 107, the binding status of the motif101 can be detected by the sensor.

The “binding status” of a binding motif, as used herein, refers towhether the binding motif is bound to a fusion molecule with acorresponding binding domain, and whether the fusion molecule is alsobound to a target molecule. Essentially, the binding status can be oneof three potential statuses: (i) the binding motif is free and not boundto a fusion molecule (see 305 in FIG. 3A); (ii) the binding motif isbound to a fusion molecule that does not bind to a target molecule (see306 in FIG. 3A); or (iii) the binding motif is bound to a fusionmolecule that is bound to a target molecule (see 307 in FIG. 3A).

Detection of the binding status of a binding motif can be carried out byvarious methods. In one aspect, by virtue of the different sizes of thebinding motif at each status, when the binding motif passes through thepore, the different sizes result in different electrical currents acrossthe pore. In one aspect, as shown in FIG. 3A, with a positive voltageapplied and KCl concentrations greater than 0.4 M in the experimentbuffer, the measured current signals 301, when 305, 306, and 307 passthrough the pore, are signals 302, 303, and 304, respectively. All threeevent types are subjected to current attenuation when KCl concentrationsare greater than 0.4 M, causing a reduction in the positive currentflow. The three signals 302, 303, and 304 can be differentiated from oneanother by the amount of the current shift (height) and/or the durationof the current shift (width), or by any other feature in the signal thatdifferentiates the three event types. It may also be that 304 iscommonly different than 302 and 303, but that 302 and 303 are notcommonly different from each other, in which case, robust detection ofthe biomarker bound to the passing molecule can still be accomplished.In another aspect, as shown in FIG. 3B, with a positive voltage appliedand KCl concentrations less than 0.4 M in the experiment buffer, themeasured current signals 308, when 312, 313, and 314 pass through thepore, are signals 309, 310, and 311, respectively. Passage of dsDNAalone causes current enhancement events (309) at KCl concentrations lessthan 0.4 M. This was shown in the published research by Smeets, Ralph MM, et al. “Salt dependence of ion transport and DNA translocationthrough solid-state nanopores.” Nano Letters 6.1 (2006): 89-95. Hence,the signal 309 can be differentiated from 310 and 311 by the eventamplitude direction (polarity) relative to the open channel baselinecurrent level (308), in addition to the three signals commonly havingdifferent amounts of the current shift (height) and/or the duration ofthe current shift (width), or by any other feature in the signal thatdifferentiates the three event types. In another aspect, as shown inFIG. 3C, with a negative voltage applied and KCl concentrations lessthan 0.4 M in the experiment buffer, the negative measured currentsignals 315, when 319, 320, and 321 pass through the pore, are signals316, 317, and 318, respectively. Compared to signals 309, 310, and 311with a positive voltage, the signals 316, 317, and 318 have the oppositepolarity since the applied voltage has the opposite (negative) polarity.In all aspects of the FIG. 3A, FIG. 3B, and FIG. 3C embodiments, thesensor comprises electrodes, which are connected to power sources andcan detect the current. Either one or both of the electrodes, therefore,serve as a “sensor.” In this embodiment, a voltage-clamp or apatch-clamp is used to simultaneously supply a voltage across the poreand measure the current through the pore.

In some aspects, an agent 106 as shown in FIG. 1 is added to the complexto aid detection. This agent is capable of binding to the targetmolecule or the ligand/target molecule complex. In one aspect, the agentincludes a charge, either negative or positive, to facilitate detection.In another aspect, the agent adds size to facilitate detection. Inanother aspect, the agent includes a detectable label, such as afluorophore.

In this context, an identification of status (iii) indicates that apolymer-fusion molecule-target molecule complex has formed. In otherwords, the target molecule is detected.

Particle Detection

The present disclosure also provides, in some aspects, methods andsystems for detecting, quantitating, and measuring particles such asproteins, protein aggregates, oligomers, or protein/DNA complexes, orcells and microorganisms, including viruses, bacteria, and cellularaggregates.

In some aspects, the pore within the structure that separates the deviceinto two volumes has a size that allows particles, such as viruses,bacteria, cells, or cellular aggregates, to pass through. A ligand thatis capable of binding to a target particle to be detected or quantitatedcan be included in the solution in the nanopore device such that theligand can bind to the unique target particle and the polymer scaffoldthrough a binding domain and a binding motif to form a complex. Manysuch particles have unique markers on their surfaces that can bespecifically recognized by a ligand. For instance, tumor cells can havetumor antigens expressed on the cell surface, and bacterial cells canhave endotoxins attached on the cell membrane.

When the formed complex in a solution loaded into the nanopore device ismoved along with the polymer scaffold to pass through the pore, thebinding status of the complex within or adjacent to the pore can bedetected such that the target microorganisms bound to the ligands can beidentified using methods similar to the molecular detection methodsdescribed elsewhere in the disclosure.

Polymer Scaffold

A polymer scaffold suitable for use in the present technology is ascaffold that can be loaded into a nanopore device and passed throughthe pore from one end to the other.

Non-limiting examples of polymers include nucleic acids, such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleicacid (PNA), dendrimers, and linearized proteins or peptides. In someaspects, the DNA or RNA can be single-stranded or double-stranded, orcan be a DNA/RNA hybrid molecule.

In one aspect, the polymer is synthetic or chemically modified. Chemicalmodification can help to stabilize the polymer, add charges to thepolymer to increase mobility, maintain linearity, or add or modify thebinding specificity. In some aspects, the chemical modification isacetylation, methylation, summolation, oxidation, phosphorylation,glycosylation, or the addition of biotin.

In some aspects, the polymer is electrically charged. DNA, RNA, PNA andproteins are typically charged under physiological conditions. Suchpolymers can be further modified to increase or decrease the carriedcharge. Other polymers can be modified to introduce charges. Charges onthe polymer can be useful for driving the polymer to pass through thepore of a nanopore device. For instance, a charged polymer can moveacross the pore by virtue of an application of voltage across the pore.

In some aspects, when charges are introduced to the polymer, the chargescan be added at the ends of the polymer. In some aspects, the chargesare spread over the polymer.

In one embodiment, each unit of the charged polymer is charged at the pHselected. In another embodiment, the charged polymer includes sufficientcharged units to be pulled into and through the pore by electrostaticforces. For example, a peptide containing sufficient entities can becharged at a selected pH (lysine, aspartic acid, glutamic acid, etc.) soas to be used in the devices and methods described herein. Likewise, aco-polymer comprising methacrylic acid and ethylene is a charged polymerfor the purposes of this invention if there is sufficient chargedcarboxylate groups of the methacrylic acid residue to be used in thedevices and methods described herein. In one embodiment, the chargedpolymer includes one or more charged units at or close to one terminusof the polymer. In another embodiment, the charged polymer includes oneor more charged units at or close to both termini of the polymer. Oneco-polymer example is a DNA wrapped around protein (e.g.DNA/nucleosome). Another example of a co-polymer is a linearized proteinconjugated to DNA at the N- and C-terminus.

Binding Motifs and Binding Domains

For nucleic acids and polypeptides such as the polymer scaffold, abinding motif can be a nucleotide or peptide sequence that isrecognizable by a binding domain. In some embodiments, the bindingdomain is a peptide sequence forming a functional portion of a protein,although the binding domain does not have to be a protein. For nucleicacids, for instance, there are proteins that specifically recognize andbind to sequences (motifs) such as promoters, enhancers, thymine-thyminedimers, and certain secondary structures such as bent nucleotide andsequences with single-strand breakage.

In some aspects, the binding motif includes a chemical modification thatcauses or facilitates recognition and binding by a binding domain. Forexample, methylated DNA sequences can be recognized by transcriptionfactors, DNA methyltransferases or methylation repair enzymes. In otherembodiments, biotin may be incorporated into, and recognized by, avidinfamily members. In such embodiments, biotin forms the binding motif andavidin or an avidin family member is the binding domain.

Molecules, in particular proteins, that are capable of specificallyrecognizing nucleotide binding motifs are known in the art. Forinstance, protein domains such as helix-turn-helix, a zinc finger, aleucine zipper, a winged helix, a winged helix turn helix, ahelix-loop-helix and an HMG-box, are known to be able to bind tonucleotide sequences.

In some aspects, the binding domains can be locked nucleic acids (LNAs),Protein Nucleic Acids of all types (e.g. bisPNAs, gamma-PNAs),transcription activator-like effector nucleases (TALENs), clusteredregularly interspaced short palindromic repeats (CRISPRs), or aptamers(e.g., DNA, RNA, protein, or combinations thereof).

In some aspects, the binding domains are one or more of DNA bindingproteins (e.g., zinc finger proteins), antibody fragments (Fab),chemically synthesized binders (e.g., PNA, LNA, TALENS, or CRISPR), or achemical modification (i.e., reactive moieties) in the synthetic polymer(e.g., thiolate, biotin, amines, carboxylates).

Target Molecule/Particles and Ligands

In the present technology, a target molecule or particle is detected orquantitated by virtue of its binding to a ligand in a fusion moleculethat binds to a polymer scaffold. A target molecule or particle and acorresponding binding ligand can recognize and bind each other. For aparticle, there can be surface molecules or markers suitable for aligand to bind (therefore the marker and the ligand form a bindingpair).

Examples of binding pairs that enable binding between a target moleculeor a molecule on a particle include, but are not limited to,antigen/antibody (or antibody fragment); hormone, neurotransmitter,cytokine, growth factor or cell recognition molecule/receptor; and ionor element/chelate agent or ion binding protein, such as a calmodulin.The binding pairs can also be single-stranded nucleic acids havingcomplementary sequences, enzymes and substrates, members of proteincomplex that bind each other, enzymes and cofactors, enzymes and one ormore of their inhibitors (allosteric or otherwise), nucleicacid/protein, or cells or proteins detectable by cysteine-constrainedpeptides.

In some embodiments, the ligand is a protein, protein scaffold, peptide,aptamer (DNA or protein), nucleic acid (DNA or RNA), antibody fragment(Fab), chemically synthesized molecule, chemically reactive functionalgroup or any other suitable structure that forms a binding pair with atarget molecule.

Therefore, any target molecule in need of detection or quantitation,such as proteins, peptides, nucleic acids, chemical compounds, ions, andelements, can find a corresponding binding ligand. For the majority ofproteins and nucleic acids, an antibody or a complementary sequence, oran aptamer can be readily prepared.

Likewise, binding ligands (such as antibodies and aptamers) can bereadily found or prepared for particles, such as protein complexes andprotein aggregates, protein/nucleic acid complexes, fragmented or fullyassembled viruses, bacteria, cells, and cellular aggregates.

Fusion Molecule

A “fusion molecule” is intended to mean a molecule or complex thatcontains two functional regions, a binding domain and a ligand. Thebinding domain is capable of binding to a binding motif on a polymerscaffold, and the ligand is capable of binding to a target molecule.

In some aspects, the fusion molecule is prepared by linking the tworegions with a bond or force. Such a bond and force can be, forinstance, a covalent bond, a hydrogen bond, an ionic bond, a metallicbond, van der Walls force, hydrophobic interaction, or planar stackinginteraction.

In some aspects, the fusion molecule, such as a fusion protein, can beexpressed as a single molecule from a recombinant coding nucleotide. Insome aspects, the fusion molecule is a natural molecule having a bindingdomain and a ligand suitable for use in the present technology.

Many options exist for connecting the binding domain with the ligand toform the fusion molecule. For example, the components may be connectedvia chemical coupling through functionalized linkers such as free amine,carboxylate coupling, thiolate, hydrazide, or azide (click) chemistry orthe binding domain and the ligand may form one continuous transcript.

FIG. 2 illustrates a more specific embodiment of the system shown inFIG. 1. In FIG. 2, the fusion molecule is a chimeric protein thatincludes a zinc finger protein or domain 202 and a humanimmunodeficiency virus (HIV) envelop protein 203. The zinc fingerprotein 202 can bind to a suitable nucleotide sequence on the polymerscaffold, a double-stranded DNA 201; the HIV envelop protein 203 canbind to an anti-HIV antibody 204 which can be present in a biologicalsample (e.g., a blood sample from a patient) for detection.

When the double-stranded DNA 201 passes through a pore 205 of a nanoporedevice 206, the nanopore device 206 can detect whether a fusion moleculeis bound to the DNA 201 and whether the bound fusion molecule binds toan anti-HIV antibody 204.

Measurement of Affinity of Binding

The present technology can be used also for measuring the bindingaffinity between two molecules and to determine other binding dynamics.For instance, after the binding motif passes through the pore of ananopore device, the device can be reconfigured to reverse the movingdirection of the polymer scaffold (as described below) such that thebinding motif can pass through the pore again.

Before the binding motif enters the pore again, one can change theconditions in the sample that is loaded into the nanopore device. Forinstance, changing the condition can be one or more of removing thetarget molecule from the sample, adding an agent that competes with thetarget molecule or the ligand for binding, and changing the pH, salt, ortemperature.

Under the changed conditions, the binding motif may be passed throughthe pore again. Therefore, whether the target molecule is still bound tothe fusion molecule can be detected to determine how the changedconditions impact the binding.

In some aspects, once the binding motif is in the pore, it is retainedthere while the conditions are changed, and thus the impact of thechanged conditions can be measured in situ.

Alternatively or in addition, the polymer scaffold can include multiplebinding motifs and each of the binding motifs can bind to a fusionmolecule that can bind to one or more specific a target molecule(s) orparticle(s). While each binding motif passes through the pore, theconditions of the sample can be changed, allowing detection of changedbinding between the ligand and the target molecule or particle on acontinued basis.

Multiplexing

In some aspects, rather than including multiple binding motifs of thesame kind as described above, a polymer scaffold can include multipletypes of binding motifs, each having different corresponding bindingdomains. In such embodiments, a sample can include multiple types offusion molecules, each type including one of the different correspondingbinding domains and a ligand for a different target molecule or targetmicroorganism.

An additional method of multiplexing includes assaying a collection ofdifferent scaffold molecules during a test, with each different scaffoldassociating with different fusion molecule(s). To determine what targetmolecules are in solution, scaffolds of the same type are labeled suchthat the sensor can identify what fusion molecule will bind to thatparticular scaffold. This can be accomplished, for example, by barcodingeach type of scaffold with polyethylene glycol molecules of varyinglengths or sizes.

With such a setting, a single polymer scaffold can be used to detectmultiple types of target molecules or target microorganisms (e.g.bacterium or virus), or target cells (e.g. circulating tumor cells).FIG. 4 illustrates such a method. Here, a double-stranded DNA 403 isused as the polymer scaffold, the double-stranded DNA 403 includingmultiple binding motifs: two copies of a first binding motif 404, twocopies of a second binding motif 405, and one copy of a third bindingmotif 406.

When the DNA passes through a nanopore device 407 that has two coaxialpores, 401 and 402, the binding status of each of the binding motifs isdetected, in which both copies of binding motif 404 bind to acorresponding target molecule, both copies of binding motif 405 bind toa corresponding target molecule; and the fusion molecule bound tobinding motif 406 does not bind to a corresponding target molecule.

This way, with a single polymer and a single nanopore device, thepresent technology can simultaneously detect multiple different targetmolecules or target microstructure (e.g., aggregates, microorganisms, orcells). Further, by determining how many copies of binding motifs arebound to the target molecules or target microorganisms, and by tuningconditions that impact the bindings, the system can obtain more detailedbinding dynamic information.

Nanopore Devices

A nanopore device, as provided, includes at least a pore that forms anopening in a structure separating an interior space of the device intotwo volumes, and at least a sensor configured to identify objects (forexample, by detecting changes in parameters indicative of objects)passing through the pore.

The pore(s) in the nanopore device are of a nano scale or micro scale.In one aspect, each pore has a size that allows a small or largemolecule or microorganism to pass. In one aspect, each pore is at leastabout 1 nm in diameter. Alternatively, each pore is at least about 2 nm,3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter.Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.

In some aspects, each pore is at least about 100 nm, 200 nm, 500 nm,1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm indiameter. In one aspect, the pore is no more than about 100000 nm indiameter. Alternatively, the pore is no more than about 50000 nm, 40000nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm,5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.

In one aspect, the pore has a diameter that is between about 1 nm andabout 100 nm, or alternatively between about 2 nm and about 80 nm, orbetween about 3 nm and about 70 nm, or between about 4 nm and about 60nm, or between about 5 nm and about 50 nm, or between about 10 nm andabout 40 nm, or between about 15 nm and about 30 nm.

In some aspects, the pore(s) in the nanopore device are of a largerscale for detecting large microorganisms or cells. In one aspect, eachpore has a size that allows a large cell or microorganism to pass. Inone aspect, each pore is at least about 100 nm in diameter.Alternatively, each pore is at least about 200 nm, 300 nm, 400 nm, 500nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm,1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2500 nm,3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm in diameter.

In one aspect, the pore is no more than about 100,000 nm in diameter.Alternatively, the pore is no more than about 90,000 nm, 80,000 nm,70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000nm, or 1000 nm in diameter.

In one aspect, the pore has a diameter that is between about 100 nm andabout 10000 nm, or alternatively between about 200 nm and about 9000 nm,or between about 300 nm and about 8000 nm, or between about 400 nm andabout 7000 nm, or between about 500 nm and about 6000 nm, or betweenabout 1000 nm and about 5000 nm, or between about 1500 nm and about 3000nm.

In some aspects, the nanopore device further includes means to move apolymer scaffold across the pore and/or means to identify objects thatpass through the pore. Further details are provided below, described inthe context of a two-pore device.

Compared to a single-pore nanopore device, a two-pore device can be moreeasily configured to provide good control of speed and direction of themovement of the polymer across the pores.

In one embodiment, the nanopore device includes a plurality of chambers,each chamber in communication with an adjacent chamber through at leastone pore. Among these pores, two pores, namely a first pore and a secondpore, are placed so as to allow at least a portion of a polymer to moveout of the first pore and into the second pore. Further, the deviceincludes a sensor capable of identifying the polymer during themovement. In one aspect, the identification entails identifyingindividual components of the polymer. In another aspect, theidentification entails identifying fusion molecules and/or targetmolecules bound to the polymer. When a single sensor is employed, thesingle sensor may include two electrodes placed at both ends of a poreto measure an ionic current across the pore. In another embodiment, thesingle sensor comprises a component other than electrodes.

In one aspect, the device includes three chambers connected through twopores. Devices with more than three chambers can be readily designed toinclude one or more additional chambers on either side of athree-chamber device, or between any two of the three chambers.Likewise, more than two pores can be included in the device to connectthe chambers.

In one aspect, there can be two or more pores between two adjacentchambers, to allow multiple polymers to move from one chamber to thenext simultaneously. Such a multi-pore design can enhance throughput ofpolymer analysis in the device.

In some aspects, the device further includes means to move a polymerfrom one chamber to another. In one aspect, the movement results inloading the polymer across both the first pore and the second pore atthe same time. In another aspect, the means further enables the movementof the polymer, through both pores, in the same direction.

For instance, in a three-chamber two-pore device (a “two-pore” device),each of the chambers can contain an electrode for connecting to a powersupply so that a separate voltage can be applied across each of thepores between the chambers.

In accordance with one embodiment of the present disclosure, provided isa device comprising an upper chamber, a middle chamber and a lowerchamber, wherein the upper chamber is in communication with the middlechamber through a first pore, and the middle chamber is in communicationwith the lower chamber through a second pore. Such a device may have anyof the dimensions or other characteristics previously disclosed in U.S.Publ. No. 2013-0233709, entitled Dual-Pore Device, which is hereinincorporated by reference in its entirety.

In some embodiments as shown in FIG. 5A, the device includes an upperchamber 505 (Chamber A), a middle chamber 504 (Chamber B), and a lowerchamber 503 (Chamber C). The chambers are separated by two separatinglayers or membranes (501 and 502) each having a separate pore (511 or512). Further, each chamber contains an electrode (521, 522 or 523) forconnecting to a power supply. The annotation of upper, middle and lowerchamber is in relative terms and does not indicate that, for instance,the upper chamber is placed above the middle or lower chamber relativeto the ground, or vice versa.

Each of the pores 511 and 512 independently has a size that allows asmall or large molecule or microorganism to pass. In one aspect, eachpore is at least about 1 nm in diameter. Alternatively, each pore is atleast about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or100 nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter.Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.

In one aspect, the pore has a diameter that is between about 1 nm andabout 100 nm, or alternatively between about 2 nm and about 80 nm, orbetween about 3 nm and about 70 nm, or between about 4 nm and about 60nm, or between about 5 nm and about 50 nm, or between about 10 nm andabout 40 nm, or between about 15 nm and about 30 nm.

In other aspects, each pore is at least about 100 nm, 200 nm, 500 nm,1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm indiameter. In one aspect, each pore is 50,000 nm to 100,000 nm indiameter. In one aspect, the pore is no more than about 100000 nm indiameter. Alternatively, the pore is no more than about 50000 nm, 40000nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm,5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.

In some aspects, the pore has a substantially round shape.“Substantially round”, as used here, refers to a shape that is at leastabout 80 or 90% in the form of a cylinder. In some embodiments, the poreis square, rectangular, triangular, oval, or hexangular in shape.

Each of the pores 511 and 512 independently has a depth (i.e., a lengthof the pore extending between two adjacent volumes). In one aspect, eachpore has a depth that is least about 0.3 nm. Alternatively, each porehas a depth that is at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm,6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm,17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60nm, 70 nm, 80 nm, or 90 nm.

In one aspect, each pore has a depth that is no more than about 100 nm.Alternatively, the depth is no more than about 95 nm, 90 nm, 85 nm, 80nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30nm, 25 nm, 20 nm, 15 nm, or 10 nm.

In one aspect, the pore has a depth that is between about 1 nm and about100 nm, or alternatively, between about 2 nm and about 80 nm, or betweenabout 3 nm and about 70 nm, or between about 4 nm and about 60 nm, orbetween about 5 nm and about 50 nm, or between about 10 nm and about 40nm, or between about 15 nm and about 30 nm.

In some aspects, the nanopore extends through a membrane. For example,the pore may be a protein channel inserted in a lipid bilayer membraneor it may be engineered by drilling, etching, or otherwise forming thepore through a solid-state substrate such as silicon dioxide, siliconnitride, grapheme, or layers formed of combinations of these or othermaterials. In some aspects, the length or depth of the nanopore issufficiently large so as to form a channel connecting two otherwiseseparate volumes. In some such aspects, the depth of each pore isgreater than 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800nm, or 900 nm. In some aspects, the depth of each pore is no more than2000 nm or 1000 nm.

In one aspect, the pores are spaced apart at a distance that is betweenabout 10 nm and about 1000 nm. In some aspects, the distance between thepores is greater than 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000nm, 7000 nm, 8000 nm, or 9000 nm. In some aspects, the pores are spacedno more than 30000 nm, 20000 nm, or 10000 nm apart. In one aspect, thedistance is at least about 10 nm, or alternatively, at least about 20nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200nm, 250 nm, or 300 nm. In another aspect, the distance is no more thanabout 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm,250 nm, 200 nm, 150 nm, or 100 nm.

In yet another aspect, the distance between the pores is between about20 nm and about 800 nm, between about 30 nm and about 700 nm, betweenabout 40 nm and about 500 nm, or between about 50 nm and about 300 nm.

The two pores can be arranged in any position so long as they allowfluid communication between the chambers and have the prescribed sizeand distance between them. In one aspect, the pores are placed so thatthere is no direct blockage between them. Still, in one aspect, thepores are substantially coaxial, as illustrated in FIG. 5A.

In one aspect, as shown in FIG. 5A, the device, through the electrodes521, 522, and 523 in the chambers 503, 504, and 505, respectively, isconnected to one or more power supplies. In some aspects, the powersupply includes a voltage-clamp or a patch-clamp, which can supply avoltage across each pore and measure the current through each poreindependently. In this respect, the power supply and the electrodeconfiguration can set the middle chamber to a common ground for bothpower supplies. In one aspect, the power supply or supplies areconfigured to apply a first voltage V₁ between the upper chamber 505(Chamber A) and the middle chamber 504 (Chamber B), and a second voltageV₂ between the middle chamber 504 and the lower chamber 503 (Chamber C).

In some aspects, the first voltage V₁ and the second voltage V₂ areindependently adjustable. In one aspect, the middle chamber is adjustedto be a ground relative to the two voltages. In one aspect, the middlechamber comprises a medium for providing conductance between each of thepores and the electrode in the middle chamber. In one aspect, the middlechamber includes a medium for providing a resistance between each of thepores and the electrode in the middle chamber. Keeping such a resistancesufficiently small relative to the nanopore resistances is useful fordecoupling the two voltages and currents across the pores, which ishelpful for the independent adjustment of the voltages.

Adjustment of the voltages can be used to control the movement ofcharged particles in the chambers. For instance, when both voltages areset in the same polarity, a properly charged particle can be moved fromthe upper chamber to the middle chamber and to the lower chamber, or theother way around, sequentially. In some aspects, when the two voltagesare set to opposite polarity, a charged particle can be moved fromeither the upper or the lower chamber to the middle chamber and keptthere.

The adjustment of the voltages in the device can be particularly usefulfor controlling the movement of a large molecule, such as a chargedpolymer, that is long enough to cross both pores at the same time. Insuch an aspect, the direction and the speed of the movement of themolecule can be controlled by the relative magnitude and polarity of thevoltages as described below.

The device can contain materials suitable for holding liquid samples, inparticular, biological samples, and/or materials suitable fornanofabrication. In one aspect, such materials include dielectricmaterials such as, but not limited to, silicon, silicon nitride, silicondioxide, graphene, carbon nanotubes, TiO₂, HfO₂, Al₂O₃, or othermetallic layers, or any combination of these materials. In some aspects,for example, a single sheet of graphene membrane of about 0.3 nm thickcan be used as the pore-bearing membrane.

Devices that are microfluidic and that house two-pore microfluidic chipimplementations can be made by a variety of means and methods. For amicrofluidic chip comprised of two parallel membranes, both membranescan be simultaneously drilled by a single beam to form two concentricpores, though using different beams on each side of the membranes isalso possible in concert with any suitable alignment technique. Ingeneral terms, the housing ensures sealed separation of Chambers A-C. Inone aspect as shown in FIG. 5B, the housing would provide minimal accessresistance between the voltage electrodes 521, 522, and 523 and thenanopores 511 and 512, to ensure that each voltage is appliedprincipally across each pore.

In one aspect, the device includes a microfluidic chip (labeled as“Dual-core chip”) is comprised of two parallel membranes connected byspacers. Each membrane contains a pore drilled by a single beam throughthe center of the membrane. Further, the device preferably has a Teflon®housing for the chip. The housing ensures sealed separation of ChambersA-C and provides minimal access resistance for the electrode to ensurethat each voltage is applied principally across each pore.

More specifically, the pore-bearing membranes can be made withtransmission electron microscopy (TEM) grids with a 5-100 nm thicksilicon, silicon nitride, or silicon dioxide windows. Spacers can beused to separate the membranes, using an insulator, such as SU-8,photoresist, PECVD oxide, ALD oxide, ALD alumina, or an evaporated metalmaterial, such as Ag, Au, or Pt, and occupying a small volume within theotherwise aqueous portion of Chamber B between the membranes. A holderis seated in an aqueous bath that is comprised of the largest volumetricfraction of Chamber B. Chambers A and C are accessible by largerdiameter channels (for low access resistance) that lead to the membraneseals.

A focused electron or ion beam can be used to drill pores through themembranes, naturally aligning them. The pores can also be sculpted(shrunk) to smaller sizes by applying a correct beam focusing to eachlayer. Any single nanopore drilling method can also be used to drill thepair of pores in the two membranes, with consideration to the drilldepth possible for a given method and the thickness of the membranes.Predrilling a micro-pore to a prescribed depth and then a nanoporethrough the remainder of the membranes is also possible to furtherrefine the membrane thickness.

In another aspect, the insertion of biological nanopores intosolid-state nanopores to form a hybrid pore can be used in either orboth pores in the two-pore method. The biological pore can increase thesensitivity of the ionic current measurements, and is useful when onlysingle-stranded polynucleotides are to be captured and controlled in thetwo-pore device, e.g., for sequencing.

By virtue of the voltages present at the pores of the device, chargedmolecules can be moved through the pores between chambers. Speed anddirection of the movement can be controlled by the magnitude andpolarity of the voltages. Further, because each of the two voltages canbe independently adjusted, the direction and speed of the movement of acharged molecule can be finely controlled in each chamber.

One example concerns a charged polymer scaffold, such as a DNA, having alength that is longer than the combined distance that includes the depthof both pores plus the distance between the two pores. For example, a1000 by dsDNA is about 340 nm in length, and would be substantiallylonger than the 40 nm spanned by two 10 nm-deep pores separated by 20nm. In a first step, the polynucleotide is loaded into either the upperor the lower chamber. By virtue of its negative charge under aphysiological condition at a pH of about 7.4, the polynucleotide can bemoved across a pore on which a voltage is applied. Therefore, in asecond step, two voltages, in the same polarity and at the same orsimilar magnitudes, are applied to the pores to move the polynucleotideacross both pores sequentially.

At about the time when the polynucleotide reaches the second pore, oneor both of the voltages can be changed. Since the distance between thetwo pores is selected to be shorter than the length of thepolynucleotide, when the polynucleotide reaches the second pore, it isalso in the first pore. A prompt change of polarity of the voltage atthe first pore, therefore, will generate a force that pulls thepolynucleotide away from the second pore as illustrated in FIG. 5C.

Assuming that the two pores have identical voltage-force influence and|V₁|=|V₂|+δV, the value δV>0 (or <0) can be adjusted for tunable motionin the V₁| (or V₂) direction. In practice, although the voltage-inducedforce at each pore will not be identical with V₁=V₂, calibrationexperiments can identify the appropriate bias voltage that will resultin equal pulling forces for a given two-pore chip; and variations aroundthat bias voltage can then be used for directional control.

If, at this point, the magnitude of the voltage-induced force at thefirst pore is less than that of the voltage-induced force at the secondpore, then the polynucleotide will continue crossing both pores towardsthe second pore, but at a lower speed. In this respect, it is readilyappreciated that the speed and direction of the movement of thepolynucleotide can be controlled by the polarities and magnitudes ofboth voltages. As will be further described below, such a fine controlof movement has broad applications.

Accordingly, in one aspect, provided is a method for controlling themovement of a charged polymer through a nanopore device. The methodentails (a) loading a sample comprising a charged polymer in one of theupper chamber, middle chamber or lower chamber of the device of any ofthe above embodiments, wherein the device is connected to one or morepower supplies for providing a first voltage between the upper chamberand the middle chamber, and a second voltage between the middle chamberand the lower chamber; (b) setting an initial first voltage and aninitial second voltage so that the polymer moves between the chambers,thereby locating the polymer across both the first and second pores; and(c) adjusting the first voltage and the second voltage so that bothvoltages generate force to pull the charged polymer away from the middlechamber (voltage-competition mode), wherein the two voltages aredifferent in magnitude, under controlled conditions, so that the chargedpolymer moves across both pores in either direction and in a controlledmanner.

To establish the voltage-competition mode in step (c), the relativeforce exerted by each voltage at each pore is to be determined for eachtwo-pore device used, and this can be done with calibration experimentsby observing the influence of different voltage values on the motion ofthe polynucleotide, which can be measured by sensing known-location anddetectable features in the polynucleotide, with examples of suchfeatures detailed later in this disclosure. If the forces are equivalentat each common voltage, for example, then using the same voltage valueat each pore (with common polarity in upper and lower chambers relativeto grounded middle chamber) creates a zero net motion in the absence ofthermal agitation (the presence and influence of Brownian motion isdiscussed below). If the forces are not equivalent at each commonvoltage, achieving equal forces involves the identification and use of alarger voltage at the pore that experiences a weaker force at the commonvoltage. Calibration for voltage-competition mode can be done for eachtwo-pore device, and for specific charged polymers or molecules whosefeatures influence the force when passing through each pore.

In one aspect, the sample containing the charged polymer is loaded intothe upper chamber and the initial first voltage is set to pull thecharged polymer from the upper chamber to the middle chamber and theinitial second voltage is set to pull the polymer from the middlechamber to the lower chamber. Likewise, the sample can be initiallyloaded into the lower chamber, and the charged polymer can be pulled tothe middle and the upper chambers.

In another aspect, the sample containing the charged polymer is loadedinto the middle chamber; the initial first voltage is set to pull thecharged polymer from the middle chamber to the upper chamber; and theinitial second voltage is set to pull the charged polymer from themiddle chamber to the lower chamber.

In one aspect, the adjusted first voltage and second voltage at step (c)are about 10 times to about 10,000 times as high, in magnitude, as thedifference/differential between the two voltages. For instance, the twovoltages can be 90 mV and 100 mV, respectively. The magnitude of the twovoltages, about 100 mV, is about 10 times of the difference/differentialbetween them, 10 mV. In some aspects, the magnitude of the voltages isat least about 15 times, 20 times, 25 times, 30 times, 35 times, 40times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times,400 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times,5000 times, 6000 times, 7000 times, 8000 times or 9000 times as high asthe difference/differential between them. In some aspects, the magnitudeof the voltages is no more than about 10000 times, 9000 times, 8000times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2000times, 1000 times, 500 times, 400 times, 300 times, 200 times, or 100times as high as the difference/differential between them.

In one aspect, real-time or on-line adjustments to the first voltage andthe second voltage at step (c) are performed by active control orfeedback control using dedicated hardware and software, at clock ratesup to hundreds of megahertz. Automated control of the first or second orboth voltages is based on feedback of the first or second or both ioniccurrent measurements.

Sensors

As discussed above, in various aspects, the nanopore device furtherincludes one or more sensors to carry out the identification of thebinding status of the binding motifs.

The sensors used in the device can be any sensor suitable foridentifying a molecule or particle, such as a polymer. For instance, asensor can be configured to identify the polymer by measuring a current,a voltage, a pH value, an optical feature, or residence time associatedwith the polymer. In other aspects, the sensor may be configured toidentify one or more individual components of the polymer or one or morecomponents bound to the polymer. The sensor may be formed of anycomponent configured to detect a change in a measurable parameter wherethe change is indicative of the polymer, a component of the polymer, orpreferably, a component bound to the polymer. In one aspect, the sensorincludes a pair of electrodes placed at two sides of a pore to measurean ionic current across the pore when a molecule or particle, inparticular a polymer, moves through the pore. In certain aspects, theionic current across the pore changes measurably when a polymer segmentpassing through the pore is bound to a fusion molecule and/or fusionmolecule-target molecule complex. Such changes in current may vary inpredictable, measurable ways corresponding with, for example, thepresence, absence, and/or size of the fusion molecules and targetmolecules present.

In one embodiment, the sensor measures an optical feature of thepolymer, a component (or unit) of the polymer, or a component bound tothe polymer. One example of such measurement includes the identificationof an absorption band unique to a particular unit by infrared (orultraviolet) spectroscopy.

When residence time measurements are used, the size of the component canbe correlated to the specific component based on the length of time ittakes to pass through the sensing device.

Still further, in embodiments directed towards detecting units of thepolymer, the sensor can include an enzyme distal to the sensing device,where the enzyme is capable of separating the terminal unit of thepolymer from the penultimate unit, thereby providing for a singlemolecular unit of the polymer. The single molecule, such as a singlenucleotide or an amino acid, can then translocate through the pore andmay or may not be detected. However, when the enzyme encounters a boundtarget molecule, the enzyme will not be able to cleave the penultimateunit, and therefore will become stalled or will skip to the nextavailable cleavage sites, thus releasing a fragment that has acomparable size difference from a single unit and is thus detectable.Detection can be done with sensors as described in this application ordetected with methods such as mass spectrometry. Methods for measuringsuch units are known in the art and include those developed by Cal Tech(see, e.g.,spectrum.ieee.org/tech-tallVat-work/test-and-measurement/a-scale-for-weighing-single-molecules).The results of such analysis can be compared to those of the sensingdevice to confirm the correctness of the analysis.

In some embodiments, the sensor is functionalized with reagents thatform distinct non-covalent bonds with each association site or eachassociated target molecule. In this respect, the gap is large enough toallow effective measuring. For instance, when a sensor is functionalizedwith reagents to detect a feature on DNA that is 5 nm on a dsDNAscaffold, a 7.5 nm gap can be used, because DNA is 2.5 nm wide.

Tunnel sensing with a functionalized sensor is termed “recognitiontunneling.” Using current technology, a Scanning Tunneling Microscope(STM) with recognition tunneling identifies a DNA base flanked by otherbases in a short DNA oligomer. As has been described, recognitiontunneling can provide a “universal reader” designed to hydrogen-bond ina unique orientation to molecules that a user desires to be detected.Most reported is the identification of nucleic acids; however, it isherein modified to be employed to detect target molecules on a scaffold.

A limitation with the conventional recognition tunneling is that it candetect only freely diffusing molecules that randomly bind in the gap, orthat happen to be in the gap during microscope motion, with no method ofexplicit capture in the gap. However, the collective drawbacks of theSTM setup can be eliminated by incorporating the recognition reagent,optimized for sensitivity, within an electrode tunneling gap in ananopore channel.

Accordingly, in one embodiment, the sensor includes surface modificationby a reagent. In one aspect, the reagent is capable of forming anon-covalent bond with an association site or an attached targetmolecule. In a particular aspect, the bond is a hydrogen bond.Non-limiting examples of the reagent include 4-mercaptobenzamide and1-H-Imidazole-2-carboxamide.

Furthermore, the methods of the present technology can provide DNAdelivery rate control for one or more recognition tunneling sites, eachpositioned in one or both of the nanopore channels, and voltage controlcan ensure that each target molecule resides in each site for asufficient duration for robust identification.

Sensors in the devices and methods of the present disclosure cancomprise gold, platinum, graphene, or carbon, or other suitablematerials. In a particular aspect, the sensor includes parts made ofgraphene. Graphene can act as a conductor and an insulator, thustunneling currents through the graphene and across the nanopore cansequence the translocating DNA.

In some embodiments, the tunnel gap has a width from about 1 nm to about20 nm. In one aspect, the width of the gap is at least about 1 nm, oralternatively, at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8,9, 10, 12, or 15 nm. In another aspect, the width of the gap is notgreater than about 20 nm, or alternatively, not greater than about 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nm. Insome aspects, the width is between about 1 nm and about 15 nm, betweenabout 1 nm and about 10 nm, between about 2 nm and about 10 nm, betweenabout 2.5 nm and about 10 nm, or between about 2.5 nm and about 5 nm.

In other embodiments, the tunnel gap is suitable for detectingmicro-sized particles (e.g., viruses, bacteria, and/or cells) and has awidth from about 1000 nm to about 100,000 nm. In some embodiments, thewidth of the gap is between about 10,000 nm and 80,000 nm or betweenabout 20,000 nm and 50,000 nm. In another embodiment, the width of thegap is between about 50,000 nm and 100,000 nm. In some embodiments, thewidth of the gap is not greater than about 100,000 nm, 90,000 nm, 80,000nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm,10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000nm, 2000 nm, or 1000 nm.

In some embodiments, the sensor is an electric sensor. In someembodiments, the sensor detects a fluorescent detection means when thetarget molecule or the detectable label passing through has a uniquefluorescent signature. A radiation source at the outlet of the pore canbe used to detect that signature.

EXAMPLES

The present technology is further defined by reference to the followingexample and experiments. It will be apparent to those skilled in the artthat many modifications may be practiced without departing from thescope of the current invention

The example section begins by first pointing out principal reasons touse a polymer scaffold and fusion molecules in biomarker detection. Aprimary reason is that a biomarker alone, below a certain sizethreshold, is undetectable with a nanopore, as shown for proteins ofvarying sizes in Calin Plesa, Stefan W. Kowalczyk, Ruben Zinsmeester,Alexander Y. Grosberg, Yitzhak Rabin, and Cees Dekker. “Fasttranslocation of proteins through solid state nanopores.” Nano letters13, no. 2 (2013): 658-663. Moreover, even those biomarkers that aredetectable may not be distinguishable. A biomarker will yield the samenanopore signature as all other molecules of comparable size/charge,preventing discrimination. By using a scaffold and fusion molecules, wecan avoid both of these problems. In particular, we show by the examplesthat detection of representative fusion molecules on scaffolds can bedemonstrated, and further that detection of target molecules to fusionmolecules on the scaffold can also be detected. With this capability,discrimination can be achieved by appropriate engineering of the liganddomain of the fusion molecule, to achieve specificity for the targetmolecule of interest.

Example 1 DNA Alone in Solid-State Nanopore Experiment

Nanopore instruments use a sensitive voltage-clamp amplifier to apply avoltage V across the pore while measuring the ionic current l₀ throughthe open pore (FIG. 6A). When a single charged molecule such as adouble-stranded DNA (dsDNA) is captured and driven through the pore byelectrophoresis (FIG. 6B), the measured current shifts from l₀ to l_(B),and the shift amount Δl=l₀−l_(B) and duration t_(D) are used tocharacterize the event. After recording many events during anexperiment, distributions of the events (FIG. 6C) are analyzed tocharacterize the corresponding molecule. In this way, nanopores providea simple, label-free, purely electrical single-molecule method forbiomolecular sensing.

In the DNA experiment shown in FIG. 6A, FIG. 6B, and FIG. 6C, the singlenanopore fabricated in silicon nitride (SiN) substrate is a 40 nmdiameter pore in 100 nm thick SiN membrane (FIG. 6A). In FIG. 6B, therepresentative current trace shows a blockade event caused by a 5.6 kbdsDNA passing in a single file manner (unfolded) through an 11 nmdiameter nanopore in 10 nm thick SiN at 200 mV and 1M KCl. The mean openchannel current is l₀=9.6 nA, with mean event amplitude l_(B)=9.1 nA,and duration t_(D)=0.064 ms. The amplitude shift is Δl=l₀−l_(B)=0.5 nA.In FIG. 6C, the scatter plot shows lΔ/l vs. t_(D) for all 1301 eventsrecorded over 16 minutes.

In the DNA experiment shown in FIG. 7A, dsDNA alone causes currentenhancement events at 100 mM KCl. This was shown in the publishedresearch of Smeets, Ralph M M, et al. “Salt dependence of ion transportand DNA translocation through solid-state nanopores.” Nano Letters 6.1(2006): 89-95). The study showed that, while the amplitude shiftΔl=l₀−l_(B)>0 for KCl concentration above 0.4 M, the shift has oppositepolarity (Δl<0) for KCl concentration below 0.4 M. As this is a negativevoltage experiment (−200 mV) with KCl concentration below 0.4 M, we seethat the DNA event has the same polarity (316) relative to the baseline(315) as shown in FIG. 3C.

Example 2 VspR Protein Binding to DNA Scaffold and Nanopore Detection

The VspR protein is a 90 kDa protein from V. cholerae that bindsdirectly to dsDNA with high micromolar affinity (see reference: Yildiz,Fitnat H., Nadia A. Dolganov, and Gary K. Schoolnik. “VpsR, a Member ofthe Response Regulators of the Two-Component Regulatory Systems, IsRequired for Expression of Biosynthesis Genes and EPSETr-AssociatedPhenotypes in Vibrio cholerae 01 El Tor.” Journal of bacteriology 183,no. 5 (2001): 1716-1726). In this example of target detection usingnanopore technology, VspR acts as the fusion molecule with asite-specific DNA binding domain, and a ligand specific binding sitethat can be engineered for the purpose of detecting a variety oftargets, including antibodies or sugars. In this demonstration, we showdetection of the VspR on the DNA scaffold as a model fusion molecule.The scaffold contains 10 VspR specific binding sites (FIG. 8). Topreserve affinity of VspR for dsDNA binding, we use 0.1 M KCl, a saltconcentration in which DNA alone translocations cause currentenhancements, as shown in Example 1 and FIG. 7A. The 5.631 kb DNAscaffold contains 10 total VspR binding sites: 5 of one sequence (14base pairs), 3 of a different sequence (18 base pairs), and 2 of a thirdsequence (27 bp). The three different sequences may not bind VspR withequal affinity. In experiments, VspR protein concentration is 18 nM inthe recording buffer, and 180 nM during labeling (binding step). Thisresults in 18× excess of VspR protein to binding sites on DNA. Theexperiment was run at pH 8.0 (pl of VspR protein is 5.8). Taking Kd andDNA concentration into account, only 0.1-1% of DNA should be fullyoccupied by VspR, with a larger percentage partially occupied, and someunknown remaining percentage of DNA entirely unbound. There is also freeVspR protein in solution during the nanopore experiment.

Two representative events are shown in FIG. 9A and FIG. 9B. In theexperiments with VspR, VspR concentration was 18 nM (1.6 mg/L), 10 nMbinding sites. The scaffold concentration was 1 nM resulting in captureevery 6.6 seconds. From this, the theoretical sensitivity using a 10 uIsample is 116 pM (0.01 mg/ml). The pore size is 15 nm in diameter andlength. The voltage is −100 mV, and note that negative voltages createnegative currents, so upward shifts correspond to attenuation events, asshown for the VspR-bound DNA event (FIG. 9B), whereas downward shiftscreate positive shifts as shown for the unbound DNA scaffold event (FIG.9A). This is consistent with the idealized signal patterns andconditions in FIG. 3C, with the DNA event (316) having faster durationand the opposite polarity compared to the fusion molecule-bound DNAevent (320). Thus, the key observation from this figure is thatVspR-bound events have the opposite signal polarity compared to unboundDNA events. FIG. 10 shows ten more representative current attenuationevents consistent with the VspR-bound scaffold passing through the pore.There were 90 such events over 10 minutes of recording, corresponding to1 VspR-bound event every 6.6 seconds. Events were attenuations of 50 to150 pA in amplitude and 0.2 to 2 milliseconds in duration. As stated,downward events correspond to current enhancement events and upwardevents correspond to current attenuation events in FIG. 9A, FIG. 9B, andFIG. 10, and this shift direction is preserved even though the baselineis zeroed for display purposes.

Example 3 RecA Protein Binding to DNA Scaffold and Nanopore Detection

RecA comprises the elements of a fusion molecule, and this exampledemonstrates the ability to use these elements to detect a targetbiomarker. Specifically, the fusion molecule consists of the portion ofRecA that binds DNA (i.e. the DNA binding domain) and the portion ofRecA (epitope) that baits the biomarker (anti-RecA antibody). DNA andRecA experiments were performed first in the absence and then in thepresence of anti-RecA antibody.

Reagent DNA/RecA consists of the 5.6 kb dsDNA scaffold molecule coatedin RecA. RecA is a 38 kDa bacterial protein involved in DNA repair,which is capable of polymerizing along dsDNA (see [C Bell. Structure andmechanism of Escherichia coli RecA ATPase. Molecular microbiology,58(2):358-366, January 2005]). This reagent is created by incubating 60nM scaffold with 112 uM RecA protein in 10 mM gamma-S-ATP, 70 mM Tris pH7.6, 10 mM MgCl, and 5 mM DTT (New England Biolabs). Gamma-S-ATP isincluded since RecA binds to dsDNA with greater affinity if the RecA hasATP bound. Since RecA can hydrolyze ATP to ADP, thereby reducing itsaffinity for DNA, the non-hydrolyzable gamma-S ATP analog prevents thistransition to ADP and thus the higher affinity state is maintained. Eventhough the ratio of RecA to DNA is one RecA molecule for every possible3-bp binding site, we expect that not all the RecA protein is bindingand thus there is free RecA in solution, as observed in other nanoporestudies (see Smeets, R. M. M., S. W. Kowalczyk, A. R. Hall, N. H.Dekker, and C. Dekker. “Translocation of RecA coated double-stranded DNAthrough solid-state nanopores.” Nano letters 9, no. 9 (2008): 3089-3095,and Kowalczyk, Stefan W., Adam R. Hall, and Cees Dekker. “Detection oflocal protein structures along DNA using solid-state nanopores.” Nanoletters 10, no. 1 (2009): 324-328). DNA/RecA samples are then adjustedto 1M KCl or LiCl, 10 mM EDTA and tested in a nanopore experiment orexcess RecA protein is removed using gel filtration (ThermoScientificSpin Columns).

In one set of experiments, we used a 16-18 nm diameter pore formed in a30 nm thick SiN membrane, applying 180 mV in 1M KCl at pH 8. In separatecontrol experiments, unbound 5.6 kb dsDNA scaffold generates 95% ofevents in the range of 2100-400 pA and 530-500 microseconds. Also, freeRecA events are 2100-600 pA, 20-200 usec. Finally, RecA-bound DNA eventsare typically much deeper blockades, in the range 0.51-3 nA, and withlonger duration (0.200-3 milliseconds). Representative events forRecA-bound DNA are shown in FIG. 11 and FIG. 12. These events haveinteresting patterns, which in the paper by Kowalczyk et al. [“Detectionof local protein structures along DNA using solid-state nanopores.” Nanoletters 10, no. 1 (2009): 324-328)] the authors attempt to infer thelocation and length of RecA filaments that are bound to each DNA;however, this is speculative, since it assumes a uniform passage ratethrough the pore even though another study showed that dsDNA does notpass through a pore at a uniform rate [Lu, Bo, et al. “Origins andconsequences of velocity fluctuations during DNA passage through ananopore.” Biophysical journal 101.1 (2011): 70-79]. FIG. 13 part (a)and (c) show on the vertical axis the maximum and mean current shift,respectively, normalized by voltage, and the event duration on thehorizontal axis. Both event plots have all 1385 events recorded over 10minutes. The amplitude is normalized by voltage to give eventconductance shift values, which is common in nanopore research papers.For example, a mean conductance of 14 nS at 200 mV is equivalent to amean current amplitude of 2.8 nA. There are two observablesub-populations in amplitude (or equivalently, conductance) andduration, with the deeper and longer duration events attributable toRecA-bound DNA and the faster shallower events attributable to free RecAin solution. We verified the identity of the faster, shallowersubpopulation as free RecA by running RecA alone control experiments.This was also verified in the earlier study [Smeets, R. M. M., S. W.Kowalczyk, A. R. Hall, N. H. Dekker, and C. Dekker. “Translocation ofRecA coated double-stranded DNA through solid-state nanopores.” Nanoletters 9, no. 9 (2008): 3089-3095]. Looking at the maximum currentshift value (FIG. 13, parts (a) and (b)) instead of the mean (FIG. 13,parts (c) and (d)) makes the subpopulations events more distinct. Notethat RecA-bound DNA vs. unbound DNA event patterns are consistent withthe model signal patterns in FIG. 3A.

In separate experiments, to demonstrate detection of a target antibody,RecA antibody was used. The DNA/RecA reagent binds an antibody biomarkercreating a DNA/RecA/Ab complex by incubating one nanomolar DNA/RecA for30 mins with either an anti-RecA monoclonal antibody (ARM191, FisherScientific) or polyclonal RecA anti-serum (gift from Prof. Ken Knight,Ph. D., UMass Medical School), at a 1:10000 dilution. Electrophoreticmobility shift assays, 5% TBE polyacrylamide gel in 1×TBE buffer, areused to test the DNA/RecA and DNA/RecA/Ab complexes by comparingmigration of complexes to DNA only or the proper controls.

The nanopore experiments were run at 200 mV in 1M LiCl with a pore thatvaried in diameter: 20 nm during the DNA alone control, and thenenlarged to 27 nm after RecA-bound DNA complexes were added. In a gelshift experiment, FIG. 14A shows a clear shift for DNA/RecA/mAb aboveDNA/RecA, which is in turn well above the unbound 5.6 kb dsDNA scaffold.This complex was tested experimentally with a nanopore. Specifically,0.1 nM DNA was added to the chamber above the pore, and after 10 minutesof recording, 1.25 nM DNA/RecA was added. After another period ofrecording, 1.25 nM DNA/RecA/mAb was added. With the AB-bound complexesin solution, a new multi-level event type was observed (FIG. 14B) thatdid not match event patterns characteristic of the other two complextypes (DNA, DNA/RecA). The Δl vs. t_(D) distributions of events recordedduring each phase of the experiment (FIG. 14C) show that RecA-bound DNAevents have longer durations t_(D), and 3 times as many events had amean amplitude shift Δl greater than 0.6 nA after DNA/RecA/mAb wasadded. A simple criteria for tagging events in this data set as alsobeing Ab-bound is (Δl, t_(D)) (0.6 nA, 0.2 ms). Identifying a bestsignature that is almost absent in unbound DNA events, but is present ina significant fraction of RecA-bound events (with or without antibodyalso bound to DNA/RecA), is useful for detection of the presence ofRecA-bound DNA complexes in solution above the nanopore. For the purposeof antibody detection, we take this a step further, and aim to identifya best signature that is almost absent in unbound DNA and RecA-bound DNAevent types, but is present in a significant fraction of RecA-boundevents with antibody also bound to DNA/RecA. This provides a criterionfor detection of the presence of RecA-bound DNA complexes in solutionabove the nanopore. As these DNA and RecA and RecA-antibody experimentsare done with a positive voltage with KCl concentration above 0.4 M, wesee that the event patterns in FIG. 14B are comparable to the idealizedpatterns in FIG. 3A.

Example 4 Fusion Molecules Comprising PNA and Biotin for Target ProteinDetection

The previous example explores detection of RecA-antibody bound toRecA-coated dsDNA complexes. Since RecA binds 3 by regionsnon-specifically, and thus RecA-antibody could bind to any RecA ondsDNA, it is also desirable to demonstrate an approach that permitstarget binding to specific sites. In particular, we use a 5.6 kb dsDNAscaffold that is engineered to bind 12-mer peptide-nucleic-acid (PNA)molecules, with each PNA having 3 biotinylated sites for binding anavidin family member (e.g., neutravidin, and or monovalent streptavidin)(FIG. 15A). The scaffold has 25 distinct sites that together localize upto 75 avidin biomarker binding sites (FIG. 15B). Our data (FIG. 16D)shows that the DNA/PNA/Neut complexes cause event signatures that aredetectable above other background event types (unbound DNA alone,Neutravidin alone, PNA/Neutravidin alone) and can therefore be tagged asfully assembled (i.e. DNA/PNA/Neutravidin) events. In this setup, it isthe fully assembled DNA/PNA/Neutravidin complex that acts as thescaffold+fusion molecule. In the remainder of the example, we providesufficient detail to show that DNA/PNA/Neutravidin complexes can bedetected with a nanopore.

In this setup, the fusion molecule contains two separate domains, onethat binds a unique DNA sequence and another that binds to ananti-Neutravidin antibody. The DNA binding domain is a protein nucleicacid molecule (PNA) that binds to the unique sequence (GAAAGTGAAAGT,uSeq1) that is repeated 25 times throughout the scaffold (FIG. 15B). PNAmolecules are similar to oligonucleotides having A, T, C, G bases, whichare capable of pairing with their complementary sequences, but insteadof a phosphate backbone like typical oligonucleotides, the backbone isprotein. This eliminates the negative charge provided by the phosphatebackbone, and thus, PNA molecules can incorporate into dsDNA bydisplacing the complementary DNA strand, making a new DNA/PNA hybrid forthe short stretch that encompasses the PNA molecule. The PNA used in theexperiment had the sequence GAA*AGT*GAA*AGT where the * indicates that abiotin was incorporated into the PNA backbone at the gamma position bycoupling to a Lysine amino acid, and thus, each PNA has three biotinmolecules (PNABio). To create the fusion molecule bound scaffold, a 60nM scaffold is heated to 95 C for 2 minutes, cooled to 60 C andincubated with a 10× excess of PNA to possible binding sites in 15 mMNaCl for 1 hr and then cooled to 4 C. The excess PNA is dialyzed out(20k MWCO, Thermo Scientific) for 2 hrs against 10 mM Tris pH 8.0. ThisDNA/PNA complex is then labeled with a 10 fold excess Neutravidinprotein (Pierce/Thermo Scientific) to possible biotin sites (assuming a60% reduction of PNA during dialysis). The reaction is electrophoresedas described above to assess purity, concentration, and potentialaggregation. This reagent, DNA/PNA/Neutravidin (D/P/N), is stored at −20C until use.

FIG. 17A and FIG. 17B show data comparing Δl vs. tD distributions fromthree separate experiments: DNA alone, Neutravidin alone, and D/P/Nreagents. The largest 1All events in the D/P/N experiment are mostlikely attributed to D/P/N complexes (FIG. 16D), providing a simplecriteria for tagging events as fusion molecule bound (i.e., scaffoldwith PNA and Neutravidin bound). Specifically, we can flag an event asbeing “fusion-molecule bound” if |Δ^(l)|>4 nA for that event. For thedata sets in FIG. 17A, 9.3% (390) of events in the D/P/N experiment have|Δ^(l)|>4 nA, with only 0.46% of D and 0.16% of N events in controlsexceeding 4 nA. In a separate experiment (data not shown) with a 7 nmdiameter pore at 1M KCl and 200 mV applied, in a control with only PNAand Neutravidin at 0.4 nM concentration, no events (0%) exceeded 4 nA.Applying our mathematical criteria, the random variable Q={Fraction offlagged events} has a binomial distribution, and using this and otherstatistical modeling tools, we can compute the 99% confidence intervalfor this data set as Q=9.29±1.15%. Since 9.29%>0.46% (the maxfalse-positive %) is satisfied well within the 99% confidence intervalfor Q, we have a positive test result, and in under 8 minutes of datagathering. In fact the same 99% confidence is achieved for this data setwith only the first 60 seconds of the data. The gel shift (FIG. 17C)shows that scaffold DNA migration is retarded in a Neutravidin dependentmanner; this guided us to using the 10× concentration in thispreliminary experiment, as it appeared all DNA is labeled and a nearlyhomogenous population is created. We do not see a shift with the DNA/PNAcomplex, though a shift was observed in another DNA/PNA nanopore studyusing shorter DNA (Alon Singer, Meni Wanunu, Will Morrison, Heiko Kuhn,Maxim Frank-Kamenetskii, and Amit Meller.” Nanopore based sequencespecific detection of duplex DNA for genomic profiling.” Nano letters10, no. 2 (2010): 738-742.).

It is to be understood that while the invention has been described inconjunction with the above embodiments, the foregoing description andexamples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1. A method for detecting a target molecule or particle suspected to bepresent in a sample, comprising: contacting the sample with (i) a fusionmolecule comprising a ligand capable of binding to the target moleculeor particle and a binding domain, and (ii) a polymer scaffold comprisingat least one binding motif to which the binding domain is capable ofbinding, under conditions that allow the target molecule or particle tobind to the ligand and the binding domain to bind to the binding motif;loading the polymer into a device comprising a pore that separates aninterior space of the device into two volumes, and configuring thedevice to pass the polymer through the pore from one volume to the othervolume, wherein the device comprises a sensor configured to identifyobjects passing through the pore; and determining, with the sensor,whether the fusion molecule bound to the binding motif is bound to thetarget molecule or particle, thereby detecting the presence of thetarget molecule or particle in the sample.
 2. The method of claim 1,wherein the target molecule is selected from the group consisting of aprotein, a peptide, a nucleic acid, a chemical compound, an ion, and anelement.
 3. The method of claim 1, wherein the target particle isselected from the group consisting of a protein complex or aggregate, aprotein/nucleic acid complex, a fragmented or fully assembled virus, abacterium, a cell, and a cellular aggregate.
 4. The method of claim 1,wherein step (a) is performed prior to step (b).
 5. The method of claim1, wherein step (b) is performed prior to step (a).
 6. The method ofclaim 1, further comprising applying a condition suspected to alter thebinding between the target molecule or particle and the ligand, andcarrying out the determination again.
 7. The method of claim 6, whereinthe condition is selected from the group consisting of removing thetarget molecule or particle from the sample, adding an agent thatcompetes with the target molecule or particle, or the ligand forbinding, and changing the pH, salt, or temperature.
 8. The method ofclaim 1, wherein the binding motif comprises a chemical modification forbinding to the binding domain.
 9. The method of claim 8, wherein thechemical modification is selected from the group consisting ofacetylation, methylation, summolation, glycosylation, phosphorylation,and oxidation.
 10. The method of claim 1, wherein the polymer is atleast one of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), apeptide nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.
 11. Themethod of claim 1, wherein the binding domain is selected from the groupconsisting of a helix-turn-helix, a zinc finger, a leucine zipper, awinged helix, a winged helix turn helix, a helix-loop-helix, and a highmobility group box (HMG-box).
 12. The method of claim 1, wherein thebinding domain is selected from the group consisting of locked nucleicacids (LNAs), peptide nucleic acids (PNAs), transcription activator-likeeffector nucleases (TALENs), clustered regularly interspaced shortpalindromic repeats (CRISPRs), dendrimers, peptides, and aptamers. 13.The method of claim 1, wherein the ligand is selected from the groupconsisting of an antibody, an antibody fragment, an epitope, a hormone,a neurotransmitter, a cytokine, a growth factor, a cell recognitionmolecule, a nucleic acid, a peptide, an aptamer, and a receptor.
 14. Themethod of claim 1, wherein the binding domain and the ligand are linkedvia an interaction selected from the group consisting of a covalentbond, a hydrogen bond, an ionic bond, a metallic bond, a van der Wallsforce, a hydrophobic interaction, and a planar stacking interaction, orare translated as a continuous polypeptide, to form the fusion molecule.15. The method of claim 1, further comprising contacting the sample witha detectable label capable of binding to the target molecule orparticle, or the target molecule or particle/ligand complex.
 16. Themethod of claim 1, wherein the polymer comprises at least two units ofthe binding motif.
 17. The method of claim 1, wherein the polymercomprises at least two different binding motifs; the sample is incontact with at least two fusion molecules each comprising a differentbinding domain capable of binding to a different one of the at least twodifferent binding motifs and a different ligand capable of binding to adifferent target molecule or particle; and the sensor is configured toidentify whether the fusion molecule bound to each binding motif isbound to a target molecule or particle.
 18. The method of claim 1,wherein the sensor comprises electrodes further configured to apply avoltage differential between the two volumes and measure current flowthrough the pore.
 19. The method of claim 1, wherein the devicecomprises an upper chamber, a middle chamber and a lower chamber,wherein the upper chamber is in communication with the middle chamberthrough a first pore, and the middle chamber is in communication withthe lower chamber through a second pore; wherein the first pore andsecond pore are about 1 nm to about 100 nm in diameter, and are about 10nm to about 1000 nm apart from each other; and wherein each of thechambers comprises an electrode for connecting to a power supply. 20.The method of claim 1, further comprising moving the polymer in areverse direction after the binding motif passes through the pore, suchas to identify, again, whether the fusion molecule bound to each bindingmotif is bound to a target molecule or particle.
 21. A kit, package ormixture for detecting the presence of a target molecule or particle,comprising: a fusion molecule comprising a ligand capable of binding tothe target molecule or particle and a binding domain; a polymer scaffoldcomprising at least one binding motif to which the binding domain iscapable of binding; and a device comprising a pore that separates aninterior space of the device into two volumes, wherein the device isconfigured to allow the polymer to pass through the pore from one volumeto the other volume, and wherein the device further comprises a sensoradjacent to the pore configured to identify whether the binding motif is(i) bound to the fusion molecule while the ligand is bound to the targetmolecule or particle, (ii) bound to the fusion molecule while the ligandis not bound to the target molecule or particle, or (iii) not bound tothe fusion molecule.
 22. The kit, package or mixture of claim 21,further comprising a sample suspected of containing the target moleculeor particle.
 23. The kit, package or mixture of claim 22, wherein thesample further comprises a detectable label capable of binding to thetarget molecule or particle, or the target molecule or particle/ligandcomplex.